RU2524629C2 - Method of identifying compound, suitable for treating inflammatory condition of oral cavity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of pharmaceutics and represents a method of identifying a compound, suitable for treatment of gingivitis, which includes an interaction of the first gingival sample, obtained from a mammal, suffering from gingivitis, with an analysed compound; an interaction of the second gingival sample from the oral cavity of a mammal with a positive control - a compound, suppressing the expression of one or more matrix metalloproteinases (halogenated diphenyl ether); measurement of a degree, with which an expression of one or more matrix metalloproteinases is suppressed by means of the analysed compound; measurement of a degree, with which the expression of one or more matrix metalloproteinases is suppressed by means of the positive control; and comparison of a degree, with which the expression of one or more matrix metalloproteinases is suppressed by means of the analysed compound, with a degree, with which the expression of one or more matrix metalloproteinases is suppressed by means of the positive control; in which the analysed compound, which suppresses the expression of one or more matrix metalloproteinases in an equal or a higher degree than the positive control, is a compound, suitable for treatment of gingivitis; where one or more matrix metalloproteinases, for which the expression is measured, include, at least, MMP-9 and MMP-13.

EFFECT: elaboration of the method of identifying the compound, suitable for treatment of gingivitis.

7 cl, 2 ex, 4 tbl, 3 dwg

Description

BACKGROUND

Periodontitis is characterized, in part, by abnormal and excessive degradation of the periodontal organic matrix. This matrix includes the gums, periodontal ligament, cementum of the teeth and alveolar bone. At least a portion of the destruction of the matrix is mediated by the super synthesis of matrix metalloproteinases (MMP), a family of zinc-dependent endopeptidases. MMPs also contribute to bone resorption by destroying osteoid tissue (i.e., demineralized and newly synthesized bone matrix), then destroying the matrix. These phenomena lead to the clinical manifestation of periodontitis, including gum atrophy, the formation of a pathological gingival pocket, loss of attachment and possible tooth loss.

SUMMARY OF THE INVENTION

The invention includes a method of treating periodontitis in a mammal in need of such treatment, comprising contacting a cell in the mouth of a mammal with an agent that suppresses at least one matrix metalloproteinase selected from the group consisting of MMP-9 and MMP-13, in which metalloproteinases are correlated with a reduction in at least one symptom associated with periodontitis.

The invention also includes a method for identifying a compound suitable for treating periodontitis in a mammal, a method comprising interacting a cell with a test compound and determining whether the test compound inhibits at least one matrix metalloproteinase selected from the group consisting of MMP-9 and MMP- 13, in which the suppression of at least one matrix metalloproteinase is an indication that the test compound is suitable for the treatment of periodontitis.

The invention also includes a method of treating periodontitis in a mammal in need of such treatment, comprising administering to the oral cavity of a mammal an oral composition containing 2,4,4'-trichloro-2'-hydroxydiphenyl ether in an amount that is effective to suppress at least , one matrix metalloproteinase in the oral cavity of a mammal, the matrix metalloproteinase selected from the group consisting of MMP-9 and MMP-13, in which the suppression of matrix metalloproteinase leads to the treatment of periodontitis mammal.

The invention further includes a method of reducing the pathological excess of the activity of matrix metalloproteinase in the oral cavity of a mammal in need of such treatment, comprising introducing into the oral cavity of a mammal an oral composition containing 2,4,4'-trichloro-2'-hydroxydiphenyl ether in an amount that is effective to reduce the activity of matrix metalloproteinase in the oral cavity of a mammal, and matrix metalloproteinase selected from the group consisting of MMP-9 and MMP-13, in which gibirovanie matrix metalloproteinase activity leads to the inhibition of excessive destruction of components of connective tissue matrix protein.

The invention includes a method for reducing the pathological excess of matrix metalloproteinase in the oral cavity of a mammal in need of such treatment, comprising introducing into the oral cavity of a mammal an oral composition containing 2,4,4'-trichloro-2'-hydroxydiphenyl ether in an amount that is effective to reduce the level of matrix metalloproteinase in the oral cavity of a mammal, in which inhibition of the level of matrix metalloproteinase leads to inhibition of excessive destruction of components connective tissue matrix protein and in which matrix metalloproteinase is selected from the group consisting of MMP-9 and MMP-13.

In one embodiment, the method comprises an oral composition comprising 0-36% by weight. silicon polishing component; 0.25-0.35% by weight of a substantially water-insoluble non-cationic antibacterial agent selected from the group consisting of halogenated diphenyl ethers, halogenated salicylanilides, benzoic esters, halogenated carbanilides and phenolic compounds; and an effective amount of 0.01-4.0% by weight. an agent that increases antibacterial activity, which improves the delivery and tight connection of the above antibacterial agent and its retention on the surface of the tooth and gums, in which the above agent that increases the antibacterial activity is (i) a copolymer of maleic acid or anhydride with another inert ethylenically unsaturated polymerizable monomer or (ii) a polymer of poly (beta-styrenephosphonic acid) or poly (alpha-styrenephosphonic acid) or a copolymer of any styrene phosphonic acid with another m ethylenically unsaturated monomer, and a composition, optionally additionally containing a source, providing an amount of fluoride ions sufficient for delivery from 25 ppm to 5000 ppm fluoride ions. In one embodiment, the oral composition comprises 0.01-36% by weight. silicon-containing polishing component. In another embodiment, the oral composition does not contain a silicon-containing polishing component.

In one embodiment, the method comprises an oral composition comprising an effective amount of anti-plaque of at least one linear water-soluble molecularly dehydrated polyphosphate salt as a necessary anti-plaque agent, an effective amount of anti-tartar, substantially water-insoluble non-cationic an antibacterial compound as a necessary anti-calculus agent, and optionally a source providing an amount of fluoride ions, is sufficient to deliver from 25 hr. / million to 5000 hr. / million fluoride ion. In one aspect, 2,4,4'-trichloro-2'-hydroxydiphenyl ether is present in the composition at a concentration of from 1 ppm to 100 ppm.

In one embodiment, the oral composition is an oral rinse or an oral rinse. In one aspect, the mouthwash or mouthwash does not contain a silicon-containing polishing agent.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the effect of 2,4,4'-trichloro-2'-hydroxydiphenyl ether on TNFα-induced MMP-9 monocyte production.

Figure 2 illustrates the effect of 2,4,4'-trichloro-2'-hydroxydiphenyl ether on PTH-induced MMP-13 osteoblast production.

Figure 3 illustrates the effect of the dentifrice of the present invention on the production of PTH induced MMP-13 in osteoblasts.

DETAILED DESCRIPTION OF THE INVENTION

The three main destructive MMPs in periodontitis are MMP-8, MMP-9, and MMP-13. MMP-8 and MMP-13 are collagenases and MMP-9 is gelatinase. All three enzymes can be found in the affected root membrane and gingival gingival fluid. The levels of these enzymes are positively correlated with the clinical signs of periodontitis. Thus, elevated or “above normal” levels of at least one of MMP-8, MMP-9, and MMP-13 are a sign of periodontitis. Measurements can be made using the enzymes MMP-8, MMP-9 and MMP-13, RNA, or biological activity.

In the present description, it is now further shown that an oral composition containing 2,4,4'-trichloro-2'-hydroxydiphenyl ether (triclosan) can be used to reduce levels of at least one of MMP-8, MMP-9, and MMP-13 in the oral cavity of a mammal. In one embodiment, the oral composition is a dentifrice. In another embodiment, the oral composition includes, for example, a mouthwash, patch, or gel. In another aspect, the antibacterial compound can be used to reduce levels of at least one of MMP-8, MMP-9, and MMP-13 in the oral cavity of a mammal.

Throughout the application, it is used that ranges are used as a symbol to describe all, without exception, quantities that are within the range. Any value within the range can be selected as the end of the range. In addition, all references cited herein are hereby incorporated by reference in their entirety. In the event of a conflict in the definitions in this disclosure and the definitions in the cited reference, this disclosure shall prevail.

Used in the present description, the term "periodontitis" refers to the improper and excessive destruction of the periodontal organic matrix, including the gums, periodontal ligament, cementum of the teeth and alveolar bone. The clinical manifestations of periodontitis include, but are not limited to, gum atrophy, the formation of a pathological gingival pocket, loss of matrix attachment, loss of tooth and bone mass. Periodontitis can be characterized as initial periodontitis, moderate periodontitis or complicated periodontitis. However, periodontitis should not be limited only to those symptoms and complications set forth in the present description, as will be obvious to a person skilled in the art. Initial periodontitis is clinically manifested, among other symptoms, by one or more of: bleeding on probing; the presence of pockets (3-4 mm); localized areas of soft tissue recession; loss of attachment (3-4 mm); bone loss (e.g. horizontal); and class I infestation of the zone of separation of the roots of multi-rooted teeth. Moderate periodontitis is clinically manifested, among other symptoms, by one or more of: the presence of pockets (4-6 mm); the presence of loss of attachment (4-6 mm); sounding bleeding; degree I and / or degree II invasion of the zone of separation of the roots of multirooted teeth; class I tooth mobility; bone loss (e.g. horizontal and / or vertical); and loss of 1/3 of the supporting alveolar bone (i.e., the ratio of crown to root is 1: 1). Complicated periodontitis is clinically manifested by one or more of: bleeding on probing; the presence of pockets (more than 6 mm); loss of attachment (more than 6 mm); degree II and / or degree III invasion of the zone of separation of the roots of multirooted teeth; class II and / or class III tooth mobility; bone loss (e.g. horizontal and / or vertical); and loss of more than 1/3 of the supporting alveolar bone (i.e., the ratio of crown to root is 2: 1 or more). Periodontitis is divided into forms, including, but not limited to: periodontitis of adults (for example, associated with plaque); early periodontitis (e.g. prepubertal, juvenile, rapidly developing and the like); periodontitis associated with systemic diseases; necrotic ulcerative periodontitis; persistent periodontitis; peri-implantitis and the like.

As used herein, the term “treatment” refers to a detectable improvement in an adverse condition and / or a reduction in symptoms of a condition after a mammal interacts with an oral composition of the invention and / or according to the method of the invention.

It should be understood that the term "treatment of periodontitis" includes preventing periodontitis in a mammal, as well as inhibiting the development of one or more pre-existing conditions associated with periodontitis in a mammal. As used herein, the terms “inhibit” and “inhibition” refer to partial inhibition or complete inhibition of periodontitis compared to an untreated condition, such as therapeutic treatment and / or prophylaxis of results. The treatment of periodontitis according to the invention, therefore, includes the reduction, inhibition, improvement, decrease, decrease, cessation or elimination of one or more of the symptoms and / or complications described in the present description.

As used herein, “pathological excess” refers to activity above an accepted normal level. For example, a “pathological excess” of matrix metalloproteinase activity is a level of matrix metalloproteinase activity that is higher than that normally found in a healthy state. As used herein, a “pathological excess of matrix metalloproteinase activity” is the level of matrix metalloproteinase activity associated with periodontitis.

As used herein, the term “suppression” refers to a decrease in enzymatic activity, a decrease in the level of enzymatic activity, a decrease in the level of the protein and / or nucleic acid encoding such a protein, or a decrease in the biochemical effect of the presence of a protein, such as one or more of MMP-8, MMP -9 and MMP-13.

In one aspect, the invention provides a method for reducing the pathological excess of at least one of MMP-8, MMP-9, and MMP-13 in the oral cavity of a mammal in need of such treatment, comprising administering to the oral cavity of a mammal an oral composition containing 2.4 , 4'-trichloro-2'-hydroxydiphenyl ether in an amount that is effective to reduce the level of matrix metalloproteinase in the oral cavity of a mammal, in which inhibition of the level of matrix metalloproteinase leads to inhibition of th destruction of components of connective tissue matrix protein.

In the present description it is stated that MMPs, such as MMP-8, MMP-9 or MMP-13, can be reduced in the oral cavity using one of many ways. In one embodiment, MMP can be reduced in the oral cavity by suppressing MMP at the nucleic acid level, as set forth elsewhere in the present description. Such a contraction may result in contraction of one or more nucleic acids encoding MMP (e.g., mRNA) and the MMP enzyme expressed in the oral cavity. Reduction of the mRNA encoding MMP, for example, can be performed using one or more of numerous methods, as will be apparent to a person skilled in the art using the disclosure set forth in the present description. Examples include the reduction of transcription of mRNA encoding MMP, and the destruction / removal of mRNA encoding MMP.

In another embodiment, MMP can be reduced in the oral cavity by directly reducing the amount of MMP enzyme. Reducing the MMP enzyme can be accomplished using one or more of numerous methods, as will be apparent to one skilled in the art using the disclosure set forth herein. Examples include, for example, inhibition of an enzyme with a small molecule inhibitor, inhibition with a natural or biologically derived molecule, proteolytic degradation of the enzyme, and affinity-based purification of the enzyme from the oral cavity. It is described herein that an agent that reduces one or more of MMP-8, MMP-9, or MMP-13 may be an agent, such as 2,4,4'-trichloro-2'-hydroxydiphenyl ether (TRICLOSAN) or it may be another antibacterial agent. In another aspect, the agent may be different from an antibacterial agent. The invention also provides methods of treating a subject affected by periodontitis.

In one aspect of the invention, the method provides a reduction in the pathological excess of matrix metalloproteinase activity in the oral cavity of a mammal in need of such treatment, comprising introducing into the oral cavity of a mammal an oral composition containing 2,4,4'-trichloro-2'-hydroxydiphenyl ether in an amount that is effective in reducing the activity of matrix metalloproteinase of at least one of MMP-8, MMP-9 and MMP-13 in the oral cavity of a mammal in which the inhibition of matrix Talloproteinases inhibit the excessive destruction of the components of the connective tissue of the matrix protein. In another aspect, the administration to the oral cavity of a mammal of an oral composition containing 2,4,4'-trichloro-2'-hydroxydiphenyl ether is carried out in an amount that is effective to reduce the level of at least one of MMP-8, MMP- 9 and MMP-13 in the oral cavity of a mammal, in which a decrease in the level of matrix metalloproteinase leads to a decrease in the complete enzymatic activity of the metalloproteinase, leading to inhibition of excessive destruction of the connective tissue components of the matrix protein. In one embodiment, a pathological excess of one or more MMPs can be reduced, as described elsewhere in this application, with respect to reducing the amount of MMPs in a mammalian oral cavity. Thus, MMP can be reduced at one or both levels of nucleic acid and protein. As described elsewhere in this application, reducing the pathological excess of one or more of these MMPs can provide a treatment for periodontitis in a mammal.

In another aspect, the invention provides a method of reducing the activity of at least one of MMP-8, MMP-9, and MMP-13 in the oral cavity of a mammal in need of such treatment, comprising administering to the oral cavity of a mammal an oral composition containing 2.4, 4'-trichloro-2'-hydroxydiphenyl ether in an amount that is effective to reduce the level of matrix metalloproteinase in the oral cavity of a mammal, in which inhibition of the level of matrix metalloproteinase leads to inhibition of excessive destruction Ia components of connective tissue matrix protein. In one embodiment, the activity of one or more MMPs can be reduced, as described elsewhere in this application, with respect to reducing the amount of MMP protein in the oral cavity of a mammal. Thus, MMP can be reduced in one or both levels of nucleic acid and protein, thereby reducing the activity of MMP in the oral cavity, or directly decreasing the activity of MMP, or indirectly reducing the level of MMP protein and / or nucleic acid.

In another aspect, the invention provides methods of treating periodontitis in a mammal in need of such treatment, comprising contacting a cell in the oral cavity of a mammal with an agent that suppresses one or both of at least one of MMP-8, MMP-9, and MMP-13. According to the invention, the suppression of metalloproteinases is correlated with a reduction of at least one symptom associated with periodontitis.

MMPs, such as MMP-8, MMP-9 or MMP-13, can be suppressed at the nucleic acid level. By way of non-limiting example, MMP can be suppressed according to the invention by suppressing mRNA encoding MMP. In one embodiment, the method of the invention comprises contacting a mammalian oral cavity with an agent that suppresses one or more of MMP-8, MMP-9, or MMP-13. This application describes that an agent that suppresses one or more of MMP-8, MMP-9, or MMP-13 may be an agent such as TRICLOSAN, or it may be another antibacterial agent. In another aspect, the agent may be different from an antibacterial agent. The invention also provides methods of treating an individual affected by periodontitis.

In another aspect, the invention provides a method of treating periodontitis in a mammal in need of such treatment. In one embodiment, a method of treating periodontitis in a mammal in need of such treatment comprises administering to the oral cavity of a mammal an oral composition containing 2,4,4'-trichloro-2'-hydroxydiphenyl ether in an amount that is effective to suppress at least one of MMP-8, MMP-9, and MMP-13, in which suppression of matrix metalloproteinase leads to treatment of periodontitis in a mammal. In another embodiment, a method of treating periodontitis in a mammal in need of such treatment comprises administering to the oral cavity of a mammal an oral composition containing 2,4,4'-trichloro-2'-hydroxydiphenyl ether in an amount that is effective to reduce the level of, at least one of MMP-8, MMP-9 and MMP-13, in which a decrease in matrix metalloproteinase leads to the treatment of periodontitis in a mammal. In yet another embodiment, a method of treating periodontitis in a mammal in need of such treatment comprises administering to the oral cavity of a mammal an oral composition containing 2,4,4'-trichloro-2'-hydroxydiphenyl ether in an amount that is effective to reduce the level of activity at least one of MMP-8, MMP-9 and MMP-13, in which a decrease in the level of matrix metalloproteinase activity leads to the treatment of periodontitis in a mammal.

In a method of treating periodontitis by administering an oral composition to the oral cavity of a mammal, the activity of one or more MMPs can be reduced, as described elsewhere in this application, with respect to reducing the amount of MMPs in the oral cavity of a mammal. Thus, MMP can be reduced at the level of one or both of the nucleic acid and protein, thus reducing the activity of MMP in the oral cavity. Similarly, the suppression of MMP or the reduction of the level of MMP can be affected by the action of the levels of either nucleic acid or protein, as described in detail elsewhere in this application.

In another aspect, the invention provides a method for identifying a compound suitable for the treatment of periodontitis in a mammal in need of such treatment, comprising contacting a cell with a test compound and determining whether the test compound inhibits one or both of at least one of MMP-8, MMP -9 and MMP-13. The suppression of at least one of the matrix metalloproteinases is an indication that the test compound is suitable for the treatment of periodontitis.

In one embodiment, a method of treating periodontitis includes administering an agent identified by the screening assay described herein, or a combination of agents that inhibit one or more periodontitis markers in which at least one of the agents is an agent identified with using the screening analysis described in this application.

In one embodiment, the invention provides a method of treating periodontitis, comprising the step of administering a therapeutically effective amount of an agent that inhibits periodontal disease and / or periodontal disorder in a subject in need of such treatment. It is determined herein that a therapeutically effective amount of an agent (i.e., an effective dosage) ranges from 0.001 to 30 mg / kg body weight, preferably from 0.01 to 25 mg / kg body weight, more preferably from 0.1 to 20 mg / kg body weight and even more preferably 1 to 10 mg / kg, 2 to 9 mg / kg, 3 to 8 mg / kg, 4 to 7 mg / kg or 5 to 6 mg / kg body weight . One of ordinary skill in the art will appreciate that certain factors can influence the dosage needed to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, general health and / or age of the subject, and other diseases present. In addition, treating a subject with a therapeutically effective amount of an inhibitor may include a single course of treatment, or preferably, may include a series of treatments. It should also be noted that the effective dosage used for treatment may increase or decrease during the course of a particular treatment. This application describes that dosage changes may result from a diagnostic study.

One skilled in the art will determine how to detect the presence of periodontitis. Additionally, one skilled in the art will determine how to identify an elevated level of one or more of MMP-8, MMP-9, and MMP-13. A representative method for detecting the presence or absence of periodontitis in a mammal involves obtaining a biological sample from the oral cavity of a test subject and interacting the biological sample with a compound or agent capable of determining one or more periodontitis markers (i.e., MMP-8, MMP-9 or MMP-13) described herein, for example, a marker nucleic acid (e.g., including mRNA, genomic DNA) or a marker peptide (e.g., including a fragment of a peptide or protein) encoded by the marker well leinovoy acid, so that the presence of the marker nucleic acids or marker peptide encoded by the nucleic acid is detected in the biological sample. In one embodiment, the agent for detecting marker mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridization with marker mRNA or genomic DNA. The nucleic acid probe may be, for example, a marker nucleic acid along the entire length or part thereof. Other suitable probes for use in diagnostic studies of the invention are described herein. In another embodiment, the activity of a periodontitis marker is used as a method for detecting a marker (i.e., the activity of MMP-8, MMP-9, or MMP-13). Any currently known or later developed study for detecting marker activity is encompassed herein.

In another embodiment, the marker peptide detection agent is an antibody capable of binding to a marker peptide, such as a detectable label antibody. Antibodies can be polyclonal or monoclonal. An intact antibody or fragment thereof can be used (e.g., Fab or F (ab '). Sub.2). It is believed that the term “labeled” with respect to a probe or antibody encompasses direct labeling of the probe or antibody by binding (ie, physically connecting) the detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by interaction with another reagent that directly tagged. Examples of indirect labeling include detecting a primary antibody using a fluorescently labeled secondary antibody and a biotin-labeled DNA probe at the end, so that it can be detected by fluorescently labeled streptavidin.

It is believed that the term “biological sample” as used herein includes tissues, cells and body fluids isolated from the oral cavity of a subject, as well as tissues, cells and fluids within the oral cavity of a subject. Thus, the detection method of the invention can be used to detect marker mRNA, peptide (eg, protein) or genomic DNA in an in vitro biological sample as well as in vivo . By way of non-limiting example, in vitro methods for detecting marker mRNA include Northern hybridization and in situ hybridization . In vitro methods for detecting marker peptides include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation and immunofluorescence. In vitro methods for detecting marker genomic DNA include Southern hybridization. In vivo methods for detecting a marker peptide include introducing a labeled anti-marker antibody into the oral cavity of a subject. For example, an antibody can be labeled with a radioactive marker, the presence of which and the location of the subject can be detected using standard image reproduction methods.

In one embodiment, the methods further include obtaining a control biological sample from a control subject, interacting the control sample with a compound or agent capable of detecting marker peptides, mRNA or genomic DNA, such that the presence of a marker peptide, mRNA or genomic DNA is detected in the biological sample, and comparison the presence of marker peptides, mRNA or genomic DNA in the control sample with the presence of marker peptides, mRNA or genomic DNA in the test sample. Alternatively, the presence of a marker peptide, mRNA, or genomic DNA in a test sample can be compared with information in a database or diagram for detection or diagnosis. In another embodiment, the methods further include the use of a control biological sample obtained from a subject having periodontitis, in which a control sample was obtained from the subject prior to the onset of periodontitis (that is, when the subject was healthy or in a “normal” state without periodontitis).

By way of a non-limiting example, the level of MMP-9 can be determined in vitro by the interaction of a cell with TNFα. In one embodiment, the cell is a monocyte. After the interaction of cells with TNFα, the MMP-9 level is detected either at the protein or nucleic acid level. In one aspect, the level of MMP-9 is also determined in vitro by the interaction of a cell with TNFα in the presence of an antibacterial agent. In one embodiment, the antibacterial agent is 2,4,4'-trichloro-2'-hydroxydiphenyl ether. In another embodiment, the agent is doxycycline. In another aspect, the agent may be different from an antibacterial agent. In one embodiment, the agent is an MMP inhibitor.

In one embodiment, the measurement of the inhibition of MMP-9 by detecting the level of MMP-9 is determined in vitro by interaction of the cell with TNFα in the presence of an agent such as an antibacterial agent, and by comparing the level of MMP-9 established in vitro by interaction of the cell with TNFα in the absence of an antibacterial agent in which the experimental conditions are otherwise identical. A lower level of MMP-9 protein, nucleic acid, or enzymatic activity in the presence of an antibacterial agent than in the absence of an antibacterial agent is an indication that the antibacterial compound inhibits MMP-9. Based on the disclosure set forth in the present description, it should be understood that the same methods can be used to evaluate MMP-8 and / or MMP-13.

It should be understood that in vitro measurement of the inhibition of MMP-8, MMP-9 and / or MMP-13 may be correlated with an in vivo effect, observation, or result. In one aspect, in vitro suppression of metalloproteinase is evidence of in vivo observation, including but not limited to treatment of periodontitis, a method for reducing pathological excess metalloproteinase and / or in vivo metalloproteinase activity, and a method for identifying a compound suitable for treating periodontitis and / or reducing a pathological excess of metalloproteinase and / or in vivo metalloproteinase activity. See, for example, Golub et al., Inflamm. Res. (1997) 46: 310-9, Preshaw et al., J. Clin. Periodontol. (2004) 31: 697-707; Mantyla et al., J. Periodontal. Res. (2003) 38: 436-439; Lorencini et al., Histol. Histopathol. (2009) 24: 157-166; and Pozo et al., J. Periodontal Res. (2005) 40: 199-207. In another aspect, in vitro suppression of metalloproteinase is a predictive parameter of an in vivo outcome, including, but not limited to, treatment of periodontitis, a method for reducing pathological excess metalloproteinase and / or in vivo metalloproteinase activity, and a method for identifying a compound suitable for treating periodontitis and / or reducing a pathological excess of metalloproteinase and / or in vivo metalloproteinase activity.

By another non-limiting example, the level of MMP-13 can be determined in vitro by the interaction of a cell with parathyroid hormone (PTH). In one embodiment, the cell is an osteoblast. After the interaction of cells with PTH, the level of MMP-13 is detected either at the level of protein or nucleic acid. In one aspect, the level of MMP-13 is also determined in vitro by the interaction of a cell with PTH in the presence of an antibacterial agent. In one embodiment, the antibacterial agent is 2,4,4'-trichloro-2'-hydroxydiphenyl ether. A lower level of MMP-13 protein, nucleic acid, or enzymatic activity in the presence of an antibacterial agent than in the absence of an antibacterial agent is an indication that the antibacterial compound inhibits MMP-13.

In one aspect, the ability of an oral composition described herein to treat periodontitis is determined by comparing the effect of 2,4,4'-trichloro-2'-hydroxydiphenyl ether in inhibiting metalloproteinase with the effect of oral composition in suppressing metalloproteinase. In another aspect, the ability of any oral composition to treat periodontitis is determined by comparing the effect of the oral composition, either in vivo or in vitro, with the effect of the oral composition described herein.

The invention further includes an oral composition, such as, for example, a dentifrice, gel, pad, mouthwash or spray for use in the method of the invention. In one aspect, the oral composition comprises an antibacterial agent. In a typical embodiment, the antibacterial agent is a non-cationic antibacterial agent. See, for example, US Pat. No. 5,288,480, which is incorporated herein by reference in its entirety. The non-cationic antibacterial agent is present in the oral composition in an effective anti-plaque amount of 0.25-0.35% by weight, preferably 0.3%. The antibacterial agent is essentially insoluble in water, implying that its solubility is less than 1% by weight. in water at 25 ° C and may even be less than 0.1%. For example, when the oral composition is an oral rinse, the concentration of the antibacterial agent can be reduced up to 10 times compared to that used in other dentifrices such as toothpaste. In one embodiment, the antibacterial agent is 2,4,4'-trichloro-2'-hydroxydiphenyl ether. In another embodiment, the oral composition comprises two or more antibacterial agents.

In one embodiment, an antibacterial activity enhancing agent (AEA) enhances the delivery of the antibacterial agent and its retention on the surfaces of the mouth. In one aspect, the AEA comprises a viscous material. See US Pat. No. 5,288,480 for descriptions of materials and compositions suitable for the AEA materials of the present invention, as well as for general descriptions of oral compositions, such as dentifrice compositions useful in the present invention. By way of a non-limiting example, the adhesive material in the composition is a polymer having a number average molecular weight of between 100,000 and 2,500,000 inclusive. In one aspect, the adhesive material is selected from polymers of polyvinylphosphonic acid, poly (1-phosphonopropene) sulfonic acid, poly (beta-styrenephosphonic acid), alpha-styrenephosphonic acid, synthetic anionic polymeric polycarboxylate, maleic anhydride, maleic acid and methyl vinyl ester. In another aspect, the adhesive molecule is a polymer of methyl vinyl ether and maleic anhydride. An agent that enhances antibacterial activity can be used at a level that is 0.01% w / w-4.0% w / w. oral composition.

As used herein, a “delivery enhancing group” refers to a group that attaches or substantially adheres, cohesively, or otherwise binds AEA (a carrier antibacterial agent) to oral (eg, tooth and gum) surfaces, thereby “delivering” an antibacterial agent to such surfaces. An organic retention enhancing group, usually hydrophobic, binds or otherwise binds the antibacterial agent to AEA, thereby facilitating the retention of the antibacterial agent with AEA and indirectly on the surfaces of the mouth. In some cases, the attachment of the antibacterial agent occurs through its physical retention using AEA, especially when the AEA is a cross-linked polymer whose structure inherently provides increased areas for such retention. The presence with a higher molecular weight of a more hydrophobic crosslinked moiety in the crosslinked polymer still further provides physical retention of the antibacterial agent to or with the aid of the crosslinked AEA polymer.

In one embodiment, an antibacterial enhancing agent that improves the delivery and tight bonding of the aforementioned antibacterial agent and its retention on the surface of the tooth and gums is (i) a copolymer of maleic acid or anhydride with another inert ethylenically unsaturated polymerizable monomer or (ii) a polymer poly (beta-styrenephosphonic acid) or poly (alpha-styrenephosphonic acid) or a copolymer of any styrenephosphonic acid with another ethylenically unsaturated monomer. However, one skilled in the art will understand that the present invention is not limited to the specific antibacterial enhancing agent used, and that other antibacterial enhancing agents are encompassed in accordance with the present invention.

In a typical dentifrice, an orally acceptable excipient comprising an aqueous phase with a moisturizing agent is present. Water is usually present in an amount of at least 3% by weight, usually 3-35%, and a moisturizing agent, preferably glycerin and / or sorbitol, usually in a total amount of 6.5-75% or 80% by weight. dentifrices, more typically 10-75%. Although not required in the present invention, in which 0.25-0.35% of a water-insoluble non-cationic antibacterial agent is optionally present, an additional ingredient may be included in the aqueous moisturizing excipient that helps solubilize the antibacterial agent in saliva. Such optional solubilizing agents include moisturizing polyols such as propylene glycol, dipropylene glycol and hexylene glycol, cellosolves such as methyl cellosolve and ethyl cellosolve, vegetable oils and waxes containing at least 12 straight-chain carbons such as olive oil, castor oil and petrol and esters such as amyl acetate, ethyl acetate and benzyl benzoate. As used herein, “propylene glycol” includes 1,2-propylene glycol and 1,3-propylene glycol. Significant amounts of polyethylene glycol should be avoided, especially with a molecular weight of 600 or more, since polyethylene glycol effectively inhibits the antibacterial activity of a non-cationic antibacterial agent. For example, polyethylene glycol (PEG) 600, when it is present with triclosan in a weight ratio of 25 triclosan: 1 PEG 600, reduces the antibacterial activity of triclosan 10-20 times the existing in the absence of polyethylene glycol.

The pH of the oral composition is usually in the range from 4.5 to 10 and in another aspect from 6.5 to 7.5. It is noteworthy that the compositions of the invention can be used orally at pH below 5, essentially without decalcification or other damage to tooth enamel. The pH can be controlled using an acid (e.g., citric acid or benzoic acid), or a base (e.g., sodium hydroxide), or a buffer solution (sodium citrate, benzoate, carbonate or bicarbonate, sodium phosphate disubstituted, sodium phosphate monosubstituted and the like).

Any abrasive solids can be used and can be selected from sodium bicarbonate, calcium phosphate (e.g., dicalcium phosphate dihydrate), calcium sulfate, precipitated calcium carbonate, silicon dioxide (e.g. hydrated silicon dioxide), iron oxide, alumina, perlite, polymer particles, for example, polyethylene, and combinations thereof. In particular, the abrasive can be selected from calcium phosphate (e.g., dicalcium phosphate dihydrate), calcium sulfate, precipitated calcium carbonate, silicon dioxide (e.g. hydrated silicon dioxide), calcium pyrophosphate, and combinations. Any type of silica may be used, such as precipitated silica or silica gel. Preferred are commercially available silicas, such as INEOS AC43, available from Ineos Silicas, Warrington, United Kingdom. Other abrasives can also be used in accordance with the present invention. US Pat. No. 4,358,437 teaches that powdered forms of calcium carbonate in abrasive form constitute one important class of such abrasive substances. Examples of these abrasives are crushed limestone or marble, calcium carbonates (chalks) such as aragonite, calcite or mixtures thereof, and synthetically precipitated calcium carbonates such as calcium carbonate in a water supply system. Typically, calcium carbonate should have a weight median diameter of less than 40 microns, preferably less than 15 microns. A second class of abrasive substances is silica powder, in particular silica-based xerogels, as defined in US Pat. No. 3,538,230.

In one embodiment, the oral composition comprises a silicon-containing polishing agent. The polishing agent may be a silicon-containing material, such as water-containing silica gel, silica-based xerogel, or a complex amorphous silica-alumina or alkali metal zirconosilicate, or precipitated silica. Colloidal silica materials include those sold under the SYLOID trademark, such as those sold as SYLOID 72 and SYLOID 74. Precipitated silicas include those sold under the ZEODENT trademark, such as ZEODENT 113, and ZEODENT 115, and ZEODENT 119.

An oral rinse or mouthwash will not typically contain abrasive particles or polishing particles. The pad will not typically contain abrasive particles or polishing particles.

Without being bound by theory, on the basis of which the advantages of this invention are achieved, it is believed that even in the absence of a special solubilizing material for an antibacterial agent (for example, 2,4,4'-trichloro-2'-hydroxydiphenyl ether), when the amount of agent is 0.25% weight.-0.35% weight. and polycarboxylate is present, a sufficient agent is present to effectively treat periodontitis by suppressing at least one of MMP-8, MMP-9 or MMP-13. This is equally applicable to the other water-insoluble non-cationic antibacterial agents described in this application.

The oral composition (for example, a dentifrice) may also contain a fluoride ion source or a fluoride providing component as an anti-caries agent in an amount sufficient to provide from 25 ppm to 5000 ppm fluoride ions. These compounds may be slightly soluble in water or may be completely soluble in water. They are characterized by their ability to release fluoride ions in water and a significant release from an undesirable reaction with other compounds of the oral drug. Among these compounds are inorganic fluoride salts, such as soluble salts of alkali metals, alkaline earth metals or, for example, sodium fluoride, potassium fluoride, ammonium fluoride, calcium fluoride, copper fluoride such as monovalent copper fluoride, zinc fluoride, barium fluoride, fluorosilicate sodium, ammonium fluorosilicate, sodium fluorozirconate, ammonium fluorozirconate, sodium monofluorophosphate, aluminum mono- and difluorophosphate and fluorinated sodium-calcium pyrophosphate. Preferred are alkali metal and tin fluorides, such as sodium and tin fluorides, sodium monofluorophosphate (MFP), and mixtures thereof. Typically, in the case of alkali metal fluorides, this component is present in an amount of not more than 2% by weight, based on the weight of the drug, and preferably in the range from 0.05% to 1%. In the case of sodium monofluorophosphate, the compound may be present in an amount of 0.1-3% and, in one embodiment, 0.7-0.8%.

In one aspect, the composition further comprises an agent selected from a tin ion agent; fluoride compounds; sodium fluoride; chlorhexidine; alexidine; hexetidine; sanguinarine; benzalkonium chloride; salicylanilide; domiphen bromide; cetylpyridinium chloride (CPC); tetradecylpyridinium chloride (TPC); N-tetradecyl-4-ethylpyridinium chloride (TDEPC); octenidine; delmopinol; octapinol; lowland; zinc ion agent; copper ion agent; essential oils; furanones; bacteriocins, ethyl lauroyl arginate, extracts of magnolia, a source of metal ions, arginine, arginine bicarbonate, gonociol, magonol, ursolic acid, ursic acid, morin, buckthorn extract, peroxide, enzyme, camellia extract, flavonoid, flavanofenine, flavanen, flavanen, flavanen, propolis.

Some embodiments of the present invention provide methods for identifying a compound suitable for treating a disease or condition of the oral cavity, a method comprising: obtaining a first gingival sample from a mammal suffering from a disease or condition of the oral cavity; obtaining a second gingival sample from the oral cavity of the above mammal; the interaction of the above first sample with the test compound; the interaction of the above second sample with a positive control, in which the above positive control is a compound known to inhibit the expression of one or more matrix metalloproteinases; measuring the degree to which the expression of one or more of the aforementioned matrix metalloproteinases is inhibited by the aforementioned test compound; measuring the degree to which the expression of one or more of the aforementioned matrix metalloproteinases is inhibited by the above positive control; and comparing the degree to which the expression of one or more of the above matrix metalloproteinases is inhibited by the above test compound, with the degree to which the expression of one or more of the above matrix metalloproteinases is suppressed by the above positive control; in which the test compound, which inhibits the expression of one or more of the aforementioned matrix metalloproteinases to an equal or greater degree than the above positive control, is a compound suitable for treating a disease or condition of the oral cavity.

In other embodiments, one or more matrix metalloproteinases is selected from the group consisting of: MMP-8, MMP-9, and MMP-13. In further embodiments, the positive control inhibits the expression of MMP-8, MMP-9, and MMP-13.

In some embodiments, the implementation of the disease or condition of the oral cavity is gingivitis or periodontitis. In other embodiments, the positive control is a halogenated diphenyl ether. In still other embodiments, the positive control is triclosan.

In some embodiments, a test compound inhibits the expression of one or more of the aforementioned matrix metalloproteinases to a greater extent than the aforementioned positive control. In additional embodiments, the test compound inhibits the expression of MMP-9 and MMP-13 to a greater extent than the above positive control. Still further embodiments provide methods in which the test compound inhibits the expression of MMP-8, MMP-9 and MMP-13 to a greater extent than the positive control.

In some embodiments, the positive control inhibits the expression of MMP-8. In some embodiments, the positive control inhibits the expression of MMP-9. In still other embodiments, a positive control inhibits the expression of MMP-13.

The invention is further described using the following examples. The examples are merely illustrative and in no way limit the scope of the invention as described and claimed.

EXAMPLES

Example 1: Obtaining MMP-9 and characterization

U937 cells and RPMI 1640 culture medium were obtained from ATCC. The human MMP-9 kit for ELISA (QUANTIKINE) was obtained from R&D Systems. Fetal bovine serum (EBS) was obtained from VWR and a solution of penicillin-streptomycin and tumor necrosis factor α (TNFα) were obtained from Sigma.

U937 human leukemia monocyte lymphoma cells were cultured in RPMI 1640 medium supplemented with 10% EBL and 1% penicillin-streptomycin solution. Cells were incubated at 37 ° C in a humidified atmosphere containing 5% CO ₂ and 95% air. Before treatment, cells were transferred to RPMI containing 1% EBS overnight. Cells were placed on a 48-well plate. The cell culture medium included either TNFα (250 ng / ml), Triclosan (1 ppm), or both agents together, or no agent (control). Cells were incubated after treatment for 24 hours. Conditioned media were collected and stored at -80 ° C until analysis. Samples of conditioned media were subjected to enzyme-linked immunosorbent assay (ELISA) for MMP-9, according to the industrial ELISA protocol (figure 1).

TNFα-stimulated U937 cells produced an increase in MMP-9. Triclosan at 1 ppm significantly reduced the level of MMP-9 in U937 cells stimulated by TNFα.

Table 1
The data for figure 1, demonstrating the effect of triclosan on the production of MMP-9 The average value (ng / ml) Standard deviation (ng / ml) the control 0.114 0,024333 TNFα 0.275 0,048665 TNFα + Triclosan 1 ppm 0.155 0,004867

Example 2: Preparation and Characterization of MMP-13

Parathyroid hormone (rat PTH 1-34) was obtained from Sigma. UMR 106-01 cells were cultured in Eagle's Minimum Support Medium (EMEM) supplemented with 25 mM Hepes pH 7.4, 1% essential amino acids, 100 units / ml penicillin, 100 μg / ml streptomycin, 5% fetal bovine serum. Real-time PCR was performed according to the following method: UMR 106-01 cells were placed in 12-well plates and cultured for 2-3 days in a cell culture medium. When the cells fused, the cell culture medium was changed to 1% fetal bovine serum overnight to grow the cells on minimal medium. Cells were preincubated with triclosan or triclosan-containing dentifrices (see, for example, US Pat. Nos. 4,894,220, 5,032,386 and related patents) for 15 minutes and then incubated with PTH (10 ^-8 M) for 4 hours.

Total RNA was isolated from UMR 106-01 cells stimulated with or without PTH with TRIzol reagent. Total RNA (0.1 μg) was reverse transcribed with cDNA using the Invitrogen SUPERSCRIPT kit according to the manufacturer's instructions. PCR was performed on cDNA using primers whose sequences are shown in Table 2. All cDNA was amplified by adding 2.5 μl of cDNA to a PCR mixture (22.5 μl) containing each primer (0.2 μM) and 12.5 μl Platinum SYBR Green qPCR SuperMix UDG (Invitrogen). The reactions were preincubated at 50 ° C for 2 minutes to purify dU-containing DNA using UDG, then at 95 ° C for 2 minutes to inactivate UDG and activate Taq. The PCR program continued with 49 cycles of denaturation at 95 ° C for 15 seconds, annealing and elongation of primers at 60 ° C for 30 seconds. The relative amount of gene expression was determined using the 2-delta delta CT method, where multiple changes in gene expression relate to control samples. All samples were normalized with respect to β-actin.

All results were expressed as mean ± standard error (S.E.) of triplicate measurements of all experiments repeated at least three times. Statistical studies were performed using t-student test.

PTH stimulated UMR cells produced an increase in MMP-13 expression. Triclosan at 10 ppm, 4 ppm and 1 ppm significantly reduced the expression of MMP-13 in PTH stimulated UMR cells. The triclosan-containing suspension of a dentifrice containing 10 ppm triclosan significantly reduced the expression of MMP-13 in PTH stimulated UMR cells.

table 2
Primer sequences Rat MMP-13 gene 5'-GCCCTATCCCTTGATGCCATT-3 '(semantic) 5'-ACAGTTCAGGCTCAACCTGCTG-3 '(antisense) Rat - actin gene 5'-AGCCATGTACGTAGCCATCC-3 '(semantic) 5'-ACCCTCATAGATGGGCACAG-3 '(antisense)

Table 3
The data for figure 2, demonstrating that triclosan inhibits the expression of MMP-13 Average value Standard deviation The control 0.558546 0.388464 PTH 78.84402 14,45422 Triclosan 10 ppm 1,518275 0.702455 Triclosan 4 ppm 1,423882 0.162156 Triclosan 1 ppm 0.416584 0.23855 Triclosan 10 ppm + PTH 3,011772 1.497531 Triclosan 4 ppm + PTH 10.55882 6,868653 Triclosan 1 ppm + PTH 35.38321 3,934183

Table 4
The data for figure 3, demonstrating that containing triclosan dentifrice inhibits the expression of MMP-13 Average value Standard deviation the control 0.917758 0,116307 PTH 370.6579 20.64484 Including triclosan suspension of dentifrice containing 10 ppm Triclosan 8,625401 5,675636

PTH + including triclosan suspension of dentifrices containing 10 ppm Triclosan 7.904052 3,23775

Claims (7)

1. A method for identifying a compound suitable for the treatment of gingivitis, including:
the interaction of the first gingival sample obtained from a mammal suffering from gingivitis, with the test compound;
the interaction of the second gingival sample obtained from the oral cavity of the above mammal with a positive control, in which the above positive control is a compound known to inhibit the expression of one or more matrix metalloproteinases;
measuring the degree to which the expression of one or more of the aforementioned matrix metalloproteinases is inhibited by the aforementioned test compound;
measuring the degree to which the expression of one or more of the aforementioned matrix metalloproteinases is inhibited by the above positive control; and
comparing the degree to which the expression of one or more of the aforementioned matrix metalloproteinases is inhibited by the above test compound with the degree to which the expression of one or more of the aforementioned matrix metalloproteinases is suppressed by the above positive control;
in which the test compound, which inhibits the expression of one or more of the above matrix metalloproteinases to an equal or greater degree than the above positive control, is a compound suitable for the treatment of gingivitis;
where the above one or more matrix metalloproteinases for which expression is measured include at least MMP-9 and MMP-13; and
where the above positive control is a halogenated diphenyl ether. 2. The method according to claim 1, in which the above one or more matrix metalloproteinases selected from the group consisting of MMP-8, MMP-9 and MMP-13. 3. The method according to claim 1 or 2, in which the above positive control inhibits the expression of MMP-8, MMP-9 and MMP-13. 4. The method according to claim 1, in which the positive control is triclosan. 5. The method according to claim 1, in which the above test compound inhibits the expression of one or more of the above matrix metalloproteinases to a greater extent than the above positive control. 6. The method according to claim 2, in which the above test compound inhibits the expression of MMP-9 and MMP-13 to a greater extent than the above positive control. 7. The method according to claim 2, in which the above test compound inhibits the expression of MMP-8, MMP-9 and MMP-13 to a greater extent than the above positive control.

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